Journal of Heredity Advance Access originally published online on June 15, 2005
Journal of Heredity 2005 96(5):582-585; doi:10.1093/jhered/esi077
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Brief Communication |
Spotting factor (Spf) from the Spotted-dilute System in Maize Is a Member of the En/Spm Controlling Element Family
From the U.S. Department of Agriculture, Agricultural Research Service, Soybean/Maize Germplasm, Pathology and Genetics Research Unit, Urbana, IL (Stinard and Sachs), and Department of Crop Sciences, University of Illinois, Urbana, IL (Sachs)
Address correspondence to Martin Sachs at Maize Genetics Cooperation Stock Center, S-108 Turner Hall, 1102 S. Goodwin Avenue, Urbana, IL 61801, or e-mail: msachs{at}uiuc.edu.
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Abstract |
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The Spotted-dilute controlling element system in maize involves an autonomous Spotting factor (Spf), and a receptor at the r1 locus haplotype R1-r(spotted dilute2). Its relationship with other maize transposable element systems is poorly characterized. Through development of a genetic tester that carries receptors for both the Spotted-dilute and the En/Spm controlling element systems, we determined that both receptors respond equally to Spf and En/Spm and that Spf is therefore a member of the En/Spm family of controlling elements.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Maize controlling elements were first described by Barbara McClintock (1950). Such systems typically involve an autonomous element that can independently excise and transpose and one or more receptor elements that are stable in the absence of the autonomous element but are capable of excision and transposition in its presence. One well-characterized maize controlling element system is Enhancer/Suppressor-mutator (En/Spm). Autonomous En or Spm elements elicit mutability at certain loci containing receptor element insertions referred to as defective Spm (dSpm) or Inhibitor (I). See Federoff (1983) and Schiefelbein et al. (1985) for a review.
The Spotted-dilute controlling element system was originally characterized as a three-element system involving the autonomous element Spf (Spotting factor), a receptor at the r1 locus in the haplotype R1-r(spotted dilute2), and a third factor called Dil (Diluting factor) that suppresses background aleurone coloration in crosses involving R1-r(spotted dilute2) (Sastry and Kurmi 1970). In the absence of Spf, kernels expressing R1-r(spotted dilute2) are pale purple. In the presence of Spf, kernels expressing R1-r(spotted dilute2) produce dark purple revertant sectors on a pale purple background. Another haplotype that responds to Spf, R1-r(spotted dilute4), has apparently been lost.
Stinard (2004b) found that Dil is an r1 haplotype-specific inhibitor of aleurone color that maps to the same location as inr1 (Stinard and Sachs 2002). Responsiveness of R1-r(spotted dilute2) to Spf is independent of the presence of Dil (Stinard 2004a). However, Dil suppresses the background aleurone color of R1-r(spotted dilute2), allowing revertant sectors to be more readily discerned.
Early tests to relate the Spotted-dilute system to known controlling element systems produced ambiguous results. Sastry and Kurmi (1970) tested Spf against the Activator Dissociation (Ac Ds; see Federoff 1983 for a review) system and found no interaction. However, preliminary data showed that lines carrying Spf induced mutability at the En/Spm receptor a2-m1::dSpm. Fincham and Sastry (1974) refer to control of R1-r(spotted dilute2) by Spm but cite unpublished results.
In contrast, Singh et al. (1975) obtained negative results when they tested Spf against c2-m2::dSpm. Gonella and Peterson (1978) found that R1-r(spotted dilute2) responds to the controlling element Fcu, but Stinard (2003) found that a variety of r1 haplotypes respond to Fcu and that this effect is independent of the Spotted-dilute system. To resolve these inconsistencies and simplify analysis of the Spotted-dilute system, tests were conducted to explore the relationship between the Spotted-dilute and En/Spm systems.
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Materials and Methods |
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Genetic Stocks
All stocks are homozygous for alleles at the anthocyanin loci necessary for purple aleurone color (A1 A2 C1 C2 R1) unless otherwise indicated. All stocks have been deposited with the Maize Genetics Cooperation Stock Center, Urbana, IL (MGSC).
R1-r(spotted dilute2) Dil Spf. Kernels from self-pollinated ears of plants heterozygous for the three factors of the Spotted-dilute controlling element system were obtained as a gift from Dr. Jerry Kermicle, University of Wisconsin (Madison). Stocks carrying these factors, separately or in combination, were derived from these starting materials by genetic crosses and self-pollinations.
dSpm/I Testers. The following lines carrying receptor elements for the En/Spm controlling element system were used in cross-pollinations to test for interactions with the autonomous element of the Spotted-dilute controlling element system, Spf: a1-m1-5719A1::dSpm sh2 (MGSC ID: 309M) and C1 sh1 bz1 wx1-m8::Spm-I8 (MGSC ID: 925F). Mutability at a1-m1-5719A1::dSpm was assayed by visual inspection of kernels for purple somatic sectors on a colorless aleurone backround. Mutability at wx1-m8::Spm-I8 was assayed either by visual inspection of kernels for starchy (translucent) revertant sectors on a waxy (opaque) endosperm background or by filing off a corner of the kernel and staining the endosperm with I2/KI solution (Coe et al. 1988). Waxy endosperm stains red with this solution, and revertant sectors, if present, stain blue.
En/Spm Autonomous Element Lines. The following lines carrying autonomous Spm controlling elements were used in cross-pollinations to determine interactions with the receptor for the Spotted-dilute system, R1-r(spotted dilute2): a1 Spm-S wx1-m8::Spm-I8 (MGSC ID: 309L) and o2-m12::Spm (MGSC ID: 703F).
R1-r(spotted dilute2) Dil wx1-m8::Spm-I8. This double tester for both Spf and En/Spm was developed by crossing an R1-r(spotted dilute2) Dil (No Spf) line to C1 sh1 bz1 wx1-m8::Spm-I8, followed by successive generations of self-pollination to homozygosity for the three factors. The recessive factors sh1 and bz1, which could render scoring for mutability at R1-r(spotted dilute2) and wx1-m8::Spm-I8 difficult, were removed by segregation and selection for Sh1 and Bz1 alleles.
Genetic Crosses
In pilot projects to find appropriate combinations of autonomous elements and receptors for both systems, crosses of lines carrying R1-r(spotted dilute2), Dil, and Spf were made to dSpm/I testers, and crosses of lines carrying Spm were made to R1-r(spotted dilute2) Dil (No Spf). Kernels from the F1 ears were planted, the resulting plants self-pollinated, and the ears scored for mutability at R1-r(spotted dilute2) and the appropriate dSpm/I tester.
To greatly increase the population of individuals scorable for mutability at both Spotted-dilute and dSpm/I receptors, a line homozygous for R1-r(spotted dilute2) Dil wx1-m8::SpmI-8 and segregating for a single Spm element was developed from crosses of R1-r(spotted dilute2) Dil to a1 Spm-S wx1-m8::Spm-I8, followed by several generations of backcrossing by R1-r(spotted dilute2) Dil wx1-m8::Spm-I8 until segregation for a single Spm element was observed as measured by 1:1 segregation for mutability at wx1-m8::Spm-I8. Multiple generations of backcrossing were required, because the a1 Spm-S wx1-m8::Spm-I8 line appears to carry more than one Spm element. Similarly, a line homozygous for R1-r(spotted dilute2) Dil wx1-m8::SpmI-8 and segregating for a single Spf element was developed from crosses of R1-r(spotted dilute2) Dil Spf to C1 sh1 bz1 wx1-m8::Spm-I8, followed by several generations of backcrossing by R1-r(spotted dilute2) Dil wx1-m8::Spm-I8 until segregation for a single Spf element was observed as measured by 1:1 segregation for mutability at R1-r(spotted dilute2). Kernels from both sets of crosses were scored for mutability at R1-r(spotted dilute2) and wx1-m8::Spm-I8.
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Results and Discussion |
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Spm Elicits Mutability at wx1-m8::SpmI-8 and R1-r(spotted dilute2)
For crosses of a1 Spm-S wx1-m8::Spm-I8 to R1-r(spotted dilute2) Dil (No Spf), eight out of nine plants showed mutability at both wx1-m8::Spm-I8 and R1-r(spotted dilute2) in the F2. One plant showed mutability at R1-r(spotted dilute2) but not at wx1-m8::Spm-I8. The possibility that the latter event represents an excision resulting in a stable null derivative of wx1-m8::Spm-I8 has not been ruled out. For crosses of o2-m12::Spm to R1-r(spotted dilute2), eight out of eight plants showed mutability at both loci in the F2.
Kernel counts from ears of plants homozygous for R1-r(spotted dilute2) Dil wx1-m8::SpmI-8 and segregating for a single Spm element are summarized in Table 1. There was nearly complete correlation of mutability at R1-r(spotted dilute2) and wx1-m8::Spm-I8 in a population of 4,776 kernels. Exceptional full colored kernels with mutable waxy endosperm indicating reversion at R1-r(spotted dilute2) (two kernels) and fully starchy kernels with mutable aleurone color indicating reversion at wx1-m8::Spm-I8 (nine kernels) were found and are also consistent with correlation between Spf and En/Spm activity. None of the revertant kernels were found to occur in sectors on the ears; therefore they most likely represent independent events. Plants grown from these exceptional kernels were crossed by homozygous R1-r(spotted dilute2) Dil wx1-m8::Spm-I8 and found to have both Spf and Spm activity.
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Spf Elicits Mutability at R1-r(spotted dilute2) and the dSpm/I receptor wx1-m8::Spm-I8
The presented data demonstrate that Spm is able to replace Spf in eliciting aleurone color mutability at R1-r(spotted dilute2). We present data indicating that Spf can replace En/Spm in eliciting mutability at wx1-m8::Spm-I8.
For crosses of R1-r(spotted dilute2) Dil Spf to a1-m1-5719A1::dSpm sh2, 14 plants were obtained that showed mutability at both R1-r(spotted dilute2) and a1-m1-5719A1::dSpm in the F2, and 5 were obtained that showed mutability at neither. There were no instances of mutability at one locus but not the other. For crosses of R1-r(spotted dilute2) Dil Spf to wx1-m8::Spm-I8, four plants were obtained that showed mutability at both R1-r(spotted dilute2) and wx1-m8::Spm-I8 in the F2 and six that showed mutability at neither. Again, there were no instances of mutability at one locus but not the other.
Kernel counts from ears of plants homozygous for R1-r(spotted dilute2) Dil wx1-m8::SpmI-8 and segregating for a single Spf element are summarized in Table 2. There was nearly complete correlation of mutability at R1-r(spotted dilute2) and wx1-m8::Spm-I8 in a population of 2,810 kernels. Exceptional full colored kernels with mutable waxy endosperm indicating reversion at R1-r(spotted dilute2) (two kernels) and fully starchy kernels with mutable aleurone color indicating reversion at wx1-m8::Spm-I8 (four kernels) were found and are also consistent with correlation between Spf and En/Spm activity. A deficit (p < .05) of mutable kernels is notable in this set of tests. This deficit could be due to reduced transmission of Spf or loss of Spf activity, but this deficit does not affect the interpretation of these results.
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These data demonstrate that Spm elicits mutability at the Spf receptor allele R1-r(spotted dilute2), and that Spf elicits mutability at the En/Spm receptor allele wx1-m8::Spm-I8. An alternative explanation is that the two systems are different but that Spm and Spf are tightly linked in the stocks used in these tests. We consider this alternative highly unlikely, because the Spf and En/Spm stocks used in these studies are unrelated, and Spf and En/Spm occur in multiple copies in the original lines and undergo transposition. It is extremely unlikely that Spf and En/Spm would always transpose together to new locations. Furthermore, we did not identify any crossovers, that is, instances of mutability at one receptor but not the other, in large populations (7,586 individuals examined in both tests). On the basis of these data, which demonstrate reciprocity of function between Spf and En/Spm in eliciting mutability at receptor alleles for both systems, we conclude that Spf is an autonomous member of the En/Spm transposable element family and that R1-r(spotted dilute2) carries a receptor element for this family.
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Acknowledgments |
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The authors' work is supported by USDA/ARS 3611-21000-019-00.
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Footnotes |
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Corresponding Editor: Susan Gabay-Laughnan
Received December 9, 2004
Accepted April 6, 2005
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References |
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